Shotgun Metagenomic Sequencing Analysis as a Diagnostic Strategy for Patients with Lower Respiratory Tract Infections

Conventional diagnostic methods (CDMs) for lower respiratory infections (LRIs) have limitations in detecting causative pathogens. This study evaluates the utility of shotgun metagenomic sequencing (SMS) as a complementary diagnostic tool using bronchoalveolar lavage (BAL) fluid. Sixteen BAL fluid samples from pneumonia patients with positive CDM results—including bacterial/fungal cultures; PCR for Mycobacterium tuberculosis or cytomegalovirus; and the BioFire® FilmArray® Pneumonia Panel (BioFire Diagnostics LLC, Salt Lake City, UT, USA)—underwent 10 Gb SMS on the Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA). Reads were aligned to the NCBI RefSeq database; with fungal identification further supported by internal transcribed spacer (ITS) analysis. Antibiotic resistance genes (ARGs) were annotated using the Comprehensive Antibiotic Resistance Database. Microbial reads accounted for 0.00002–0.04971% per sample. SMS detected corresponding bacteria in 63% of cases, increasing to 69% when subdominant taxa were included. Fungal reads were low; however, Candida species were identified in four samples via ITS. No viral reads were detected. ARGs meeting perfect match criteria were found in two cases. This is the first real-world study comparing SMS with CDMs, including semiquantitative PCR, in BAL fluid for LRI. SMS shows promise as a supplementary diagnostic method, with further research needed to optimize its performance and cost-effectiveness.